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IN MILE 1 DIOBU AREA OF PORT HARCOURT, NIGERIA

Obire, Omokaro* and Berembo, Beremboba Telema

Department of Applied and Environmental Biology, Rivers State

University Of Science and Technology, P.M.B. 5080, Port Harcourt, Nigeria,

*Correspondence Author E–Mail: [email protected]

ABSTRACT

The microbiology of three different yoghurt samples from Mile I Diobu area of Port

Harcourt was evaluated weekly for three weeks using standard plate count and most

probable number (MPN) technique. This was carried out by analyzing for total

aerobic heterotropic bacteria, total coliform bacteria, Thermotolerant coliform

bacteria and fungi. The total aerobic heterotrophic bacteria count ranged from 4.0 ×

105cfuml

-1 to 1.13 × 10

6cfuml

-1 of yoghurt, the total coliform bacteria ranged from 11

to 140 coliform (MPN) 100ml-1

while the thermotolerant coliform bacteria ranged

from 17 to 90 coliform (MPN) 100ml-1

. The fungal count ranged from 1.0 × 102 spore

forming unit (sfu) ml-1

to 5.0 × 102

sfuml-1

. The results of the mean values of pH of

the samples were Green field yoghurt (pH 7.0), Home victory yoghurt (pH 7.5), and

Mary gold natural yoghurt (pH 5.0). Generally, the bacterial, fungal and

thermotolerant coliform counts were highest in the Mary gold samples which had an

acidic pH. This shows that the isolates are acidophiles. On the other hand, the

bacterial and fungal counts were lowest in Green field samples with a neutral pH

which however, recorded the highest total coliform count. While the total coliform

and thermotolerant coliform counts were lowest in the Home victory yoghurt samples.

Generally, the incidence (%) of bacteria was; Bacillus cereus (22.5%),

Bifidobacterium sp (7.5%), Escherichia coli (7.5%), Lactobacillus acidophilus (15%),

Lactobacillus bulgaricus (12.5%), Pseudomonas aeruginosa (15%), and

Staphylococcus aureus (20.0%). Incidence of fungi was; Aspergillus niger (10%),

Fusarium solani (15%), Mucor sp (20%), Penicillium italicum and Penicillium spp

(35%), and Saccharomyces cerevisae (20%). Statistical analysis using ANOVA

showed that there is no significant difference at P = 0.05 in the microbial counts and

in the incidence of the bacterial isolates between the three yoghurt samples. The

presence of these bacteria and fungi especially enteric organisms and indicators of

faecal contamination such as E. coli and Enterobacter is of public health concern as

they pose serious health hazards to the unsuspecting consumers.

Key words: Yoghurt, bacteria, fungi, faecal coliform. E. coli

INTRODUCTION

Yoghurt is a soured milk product known for ages. It is a custard-like food with a tart

flavor prepared from milk curdled by bacteria especially Lactobacillus bulgaricus and

Streptococcus thermophilus and often sweetened or flavoured with fruit (American

heritage, 2000). The L. bulgaricus produces amino acids which stimulate S.

thermophilus to produce formic acid which is essential for the growth and survival of

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(5), 9, 2014 180

the L. bulgaricus. The S. thermophilus turns the milk sour while L. bulgaricus

produces the typical yoghurt aroma. Yoghurt can be made from the milk of goat, cow,

ewe and buffalo or a combination of these milk (Alderton et al., 2000).

Yoghurt is low in saturated fat and cholesterol but nutritionally rich in Protein,

vitamins including Pantothenic acid, and Riboflavin. It is also a very good source of

calcium, iron, potassium, other minerals and phosphorus which maintains the Red

blood cells and helps keep your nervous system functioning well (Korlar and Aowi,

1994). Yoghurt may prevent high blood pressure. The potassium in yoghurt almost

600mg per eight ounce may help flush some of the excess sodium out of our body.

The protein, carbohydrate and vitamin content are higher in yoghurt than in milk

(Porter and Dryden, 1998; Parnel et al., 2006). There is a little different between milk

and yoghurt in terms of energy content, but sweetened yoghurt is richer in energy

sources than milk (Dryden, 1999).

Yoghurt has an antimicrobial activity to some bacteria (Hingst, 2000). The lactic acid

found in yoghurt also helps to protect your gum and hinder protein putrefaction in the

intestine (Schulz and Hingst, 2000). Yoghurt also has a nutritional benefit beyond that

of milk, because lactose intolerant individual sometimes tolerate yoghurt better than

other dairy products. The starter culture produces a lactose enzyme that aids the

digestion (Shukla and Leifson, 2002). Consumption of yoghurt helps to alter

microbial flora of the intestine. Yoghurt contains probiotics, beneficial bugs that helps

crowd out harmful micro-organisms that can cause intestinal infections (Amanda et

al., 2013).

Types of commercially made yoghurts are powdered yoghurt, soft or liquid yoghurt

and firm yoghurt. The most popular type commonly produced is firm yoghurt

(Hardman and Milliken, 1998). Microorganisms can contaminate yoghurt through

different steps associated with its production. Fresh milk used in preparation may

contain resistant spores of Bacillus and Clostridium species (Jay et al., 1999). The

addition of fruit, flavour, and sugar into yoghurt may act as a means to introduce yeast

and moulds into the product. Yeast contaminant gives off flavours, loss of texture

quality and eventually swelling and blowing of the container (Alderton, 2000).

Contaminants may get into the yoghurt during dispensation if proper good

manufacturing practice (GMP) is not put into place during the process of production.

The aim and scope of this study is to determine the standard plate count of total

bacteria and fungi of yoghurt samples, to estimate the total coliform and

thermotolerant coliform bacteria using the most probable number technique (MPN

technique); to isolate, characterize and determine the incidence of bacteria and fungi

in samples of yoghurt as to ascertain the microbial load, pathogenic microorganisms

present if any and to ascertain the sanitary level of the yoghurt producers or handlers.

MATERIALS AND METHOD

Collection of Yoghurt Samples

Samples of three different brands of yoghurt packaged in plastic bottles were bought

from a distributor at Emenike Street in Mile 1 Area of Port Harcourt. The brands

were Green Field yoghurt, Home Victory yoghurt, and Mary Gold natural yoghurt.

Green field yoghurt is produced in Eleme; ingredients are Skimmed Milk, Sugar,

yogflex starter culture and treated water. Home victory yoghurt is produced in Amadi-

Ama; ingredients are Full cream milk, sugar, yogflex starter culture and treated water



while Mary Gold natural yoghurt is produced at Elelenwo; ingredient are Fresh milk,

premium water, sugar, and flavour.

The samples were bought in frozen state and put into ice packed containers and

immediately transported to the laboratory for analysis. The microbiological analyses

were conducted after the frozen yoghurt samples were allowed to thaw and before the

expiry dates of the products in July, August and September, 2013.

Determination of the pH

The pH of each yoghurt sample was determined by using Jenway pH meter. The

sterilized pH rod of the meter was inserted into a beaker of distilled water for

standardization. Each thawed yoghurt sample was thoroughly mixed and poured into

sterile beaker after which the pH rod was inserted into the sample and reading was

recorded after the readings have stabilized on the screen of the meter. This process

was repeated for each yoghurt sample used during this study.

Cultivation and Enumeration of Total Heterotrophic Bacteria and Fungi

Enumeration of Viable Microbial count of microorganisms, the total viable count of

bacteria and fungi in the yoghurt samples were estimated using the spread plate

method.

Serial dilution was carried out on each yoghurt sample. The dilution factor for the

isolation of bacteria was 10-5

while the dilution factor for the isolation of fungi was

10-2

. This was done so as to obtain discrete colonics when plated on the medium. One

milliliter (1.0ml) of each yoghurt sample was added to separate 9.0ml of normal saline

(diluent) and further dilution was made up to 10-5

and 10-2

.

An aliquot (0.1ml) of the appropriately diluted sample was then inoculated onto

nutrient agar plates for the isolation of bacteria and onto Sabouraud dextrose agar

plates for the isolation of fungi. The spread plate method was done using sterile bent

glass spreader to spread the sample evenly on the agar plates. Cultures were prepared

in duplicates. Cultured Nutrient agar plates were incubated at 370C for 24 hours while

the cultured SDA plates were incubated on the laboratory bench for 3 to 5 days.

Discrete colonies that developed on the plates (overnight culture) were counted, the

average taken and recorded as total heterotrophic counts of bacteria.

Discrete colonies were collected aseptically and streaked onto nutrient agar plates (for

bacteria purification) and incubated at 370C overnight. Pure colonies were later stored

in Mac Cartney bottles containing nutrient agar slants and put into the fridge as stocks

cultures for further biochemical tests. A total of eleven (11) pure cultures were stored

and regarded as the bacteria isolates. Colonies which developed after 5 days on SDA

plates were counted and the average count for the duplicate cultures were recorded as

total viable fungi of each sample. The colour and colonial morphologies or

characteristics were also recorded. Discrete colonies were subcultured onto freshly

prepared SDA to obtain pure cultures.

Estimation of Coliforms

Estimation of the coliform bacteria was done using the most probable number

technique (MPN technique). Reaction to MPN technique and thermotolerant coliform

bacteria MPN index 100ml of each yoghurt sample was done using double strength

MacConkey broth for 10ml of sample and single strength MacConkey broth for 0.1ml

and 1ml of the sample. The test for the estimation of coliforms involves the following


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steps: presumptive, confirmatory and completed test. It was performed as described by

Verma et al., (1999).

Enumeration of Faecal Coliform Test

The test for coliform does not distinguish coliform of animal origin and from others

(Doyle and Erickson, 2006). In this test, the test tube with the production of gas in the

presumptive test were streaked with the aid of a sterile wire loop onto MacConkey

agar plates, and incubated at 370C for 24 hours.

Isolation, Characterization and Identification of Bacteria in Yoghurt Samples

Pure cultures of bacteria were obtained by aseptically streaking representative

colonies of different morphological types which appeared on the cultured plates onto

freshly prepared nutrient agar plates which were incubated at 280C for 24 hours. The

isolates which developed were further sub cultured onto agar slopes/slants and

incubated at 280C for 24 hours. These served as pure stock cultures used for

subsequent characterization tests. The following characterization tests were performed

in duplicates. Gram staining, catalase test, coagulase test, urease test sugar

fermentation test, methyl red test, indole test and acid gas test were carried out as

described by Cappuccino and Macfaddin (2005) and Kirk et al., (2005). The pure

cultures were identified on the basis of their cultural, morphological and physiological

characteristics in accordance with methods described by Cruikshank et al., (1975) and

with reference to Holt (1977).

Isolation, Characterization and Identification of Fungi in Yoghurt Samples

Pure cultures of fungi were obtained by sub culturing discrete colonies onto freshly

prepared Sabouraud dextrose agar plates and incubated at 280C for 5 to 7 days. The

colonies which developed were further subcultured onto agar slopes or slants and

incubated at 280C for 5 to 7 days. The following standard characterization tests were

performed in duplicate; macroscopic examination of fungal growth was carried out by

observing the colony morphology-diameter, colour (pigmentation), texture and surface

appearance. Microscopic examination was done by needle mount or wet mount

method and observing sexual and asexual reproductive structures.

Microscopic examination of fungi

A wet mount was carried out for the fungi isolated. A drop of sterile distilled water

was aseptically dropped on a grease free clean slide. A piece of fungal hyphae under

test was teased into it using two sterile needles. The teasing was done carefully and

slowly so as to make good spread of the fungal hyphae. Each prepared slide was gently

covered with a cover slip to avoid air bubble. The slides were observed under low and

high power objective, and observation recorded as the cultural characteristics,

sporangia, conidia, arthrospores, and vegetative mycelium, septate and non-septate

hyphae according to Barnett and Hunter (1972).

RESULTS

Total Viable Count for bacteria and fungi of the different yoghurt samples

The results of the mean values of pH of the yoghurt samples were Green field yoghurt

(pH 7.0), Home victory yoghurt (pH 7.5), and Mary Gold natural yoghurt (pH 5.0).



The result of the mean value of total viable count for bacteria and fungi of the

different yoghurt samples is shown in Table 1 and in Table 2 respectively.

The mean values of the total viable bacteria of Green field yoghurt, Home victory

yoghurt, and Mary Gold natural yoghurt samples ranged from 4.0 × 105 to 5.2 x

105cfuml

-1, 4.0 × 10

5 to 5.5 × 10

5cfuml

-1, and 6.0 × 10

5 to 1.13 × 10

6cfuml

-1

respectively. While the mean value of the total fungal count ranged from 1.0 × 102 to

3.0 × 102cfuml

-1, from 2.0 × 10

2 to 5.0 × 10

2cfuml

-1, and from 3.0 × 10

2 to 5.0 ×

102cfuml

-1 respectively.

Generally, both bacterial and fungal counts were highest in

Mary Gold natural yoghurt samples and lowest in Green field yoghurt.

The result of the total coliform and of the thermotolerant coliform and facecal coliform

is shown in Table 1 and Table 2 respectively. The total coliform count ranged from 11

to 140 coliform (MPN) 100ml-1

while the thermotolerant coliform and facecal

coliform ranged from 17 to 90 coliform (MPN) 100ml-1

of yoghurt sample.

The incidence (%) of bacteria isolated from each yoghurt sample is shown in Table 3.

Generally, incidence of bacteria in all the samples of yoghurt were; Bacillus cereus

(22.5%), Bifidobacterium sp (7.5%), Escherichia coli (7.5%), Lactobacillus

acidophilus (15%), Lactobacillus bulgaricus (12.5%), Pseudomonas aeruginosa

(15%), and Staphylococcus aureus (20.0%). However, Bifidobacterium sp,

Escherichia coli, and Lactobacillus bulgaricus were not isolated from Green field

yoghurt, Home victory yoghurt and Mary Gold natural yoghurt respectively.

The incidence fungi isolated from each yoghurt sample is shown in Table 4.

Generally, the fungi isolated and incidence (%) was Aspergillus niger (10%),

Fusarium solani (15%), Mucor sp (20%), Penicillium italicum and Penicillium sp

(35%) and Saccharomyces cerevisiae (20%). All the fungi were isolated from Mary

gold natural yoghurt while Mucor and Penicillium species were not isolated from

Green field yoghurt and Home victory yoghurt respectively.

Statistical analysis using ANOVA showed that the Calculated F- value for the data

obtained for the microbial counts and for the incidence of the bacterial isolates was

2.85 and 0.12 respectively. These F – values are lower than their respective tabular

values at P = 0.05. This showed that, there is no significant difference at P = 0.05 in

the microbial counts and in the incidence of the bacterial isolates between the three

yoghurt samples.

Table 1: Total coliform bacteria Count of various yoghurt samples

Yoghurt sample Total Coliform Bacteria (MPN) INDEX/100ml of yoghurt

Week 1 Week 2 Week 3

Green field

140 70 17

Home victory

17 11 26

Mary Gold

Natural

70 26 70


(5), 9, 2014 184

Table 2: Thermotolerant Coliform Bacteria and Facecal Coliform Bacteria

Count of the various yoghurt samples

Yoghurt sample Thermotolerant Coliform Bacteria and Facecal

Coliform Bacteria ( MPN) INDEX/100ml of yoghurt

Week 1 Week 2 Week 3

Green field

33 33 33

Home victory

17 17 17

Mary Gold Natural 90 90 90

Table 3: Incidence (%) of Bacteria Isolated From Each Yoghurt Sample

Isolates Green field

yoghurt

Home Victory

yoghurt

Mary Gold

natural yoghurt

Bacillus cereus 16.67 21.43 28.57

Bifidobacterium sp - 7.14 14.29

Escherichia coli 16.67 - 7.14

Lactobacillus acidophilus 16.67 14.29 14.29

Lactobacillus bulgaricus 16.67 21.43 -

Pseudomonas aeruginosa 25 14.29 7.14

Staphylococcus aureus 8.33 21.43 28.57

Table 4: Incidence (%) of Fungi Isolated From Each Yoghurt Sample

Fungi Green field

yoghurt

Home Victory

yoghurt

Mary Gold

natural yoghurt

Aspergillus niger 16.67 - 14.29

Fusarium solani 16.67 14.29 14.29

Mucor sp - 28.57 28.57

Penicillium

italicum

33.33 14.29 14.29

Penicillium sp 16.67 14.29 14.29

Saccharomyces

cerevisae

16.67 28.57 14.29

DISCUSSION

The present study has revealed the population and types of bacteria, fungi and of

coliforms in the various samples of yoghurt. The results of the mean values of pH of

the samples were Green field yoghurt was in the neutral range, Home victory yoghurt

is slightly alkaline and Mary gold natural yoghurt is acidic. Generally, the bacterial,



fungal and thermotolerant coliform counts were highest in the Mary gold samples

which had an acidic pH. This shows that the isolates are acidophiles. It has also been

reported that yoghurt that has an acidic content seem to act as a selective media for

yeasts and moulds using lacteal as their possible source of energy (Porter et al., 2005).

On the other hand, the bacterial and fungal counts were lowest in Green field samples

with a neutral pH which however, recorded the highest total coliform count. While the

total coliform and thermotolerant coliform counts were lowest in the Home victory

yoghurt sample which is slightly alkaline.

The presence of various types of bacteria and fungi was also revealed. Statistical

analysis showed that there was no significant difference at P = 0.05 in the microbial

counts and in the incidence of the bacterial isolates between the three yoghurt

samples. Among the bacteria isolates Bacillus cereus had the highest incidence of

22.5% while; Bifidobacterium sp and Escherichia coli recorded the lowest incidence

of 7.5% each. Among the fungi isolates Penicillium italicum and Penicillium spp had

the highest incidence of 35% while Aspergillus niger recorded the lowest incidence of

10%. Bacteria such as Lactobacillus acidophilus, Lactobacillus bulgaricus, and

Bifidobacterium bifidum isolated in this present study has been reported by Suaze et

al., (2000) as beneficial microorganisms found in yoghurt. These organisms which are

the starter culture for the fermentation of milk to produce yoghurt have been termed

legal milk bacteria (Eka and Ohaba, 1997). Escherichia coli and Staphylococcus

aureus isolated in this study has been reported and proved to be potential

contaminants of yoghurt (David and Carr, 2003). The incidence of Staphylococcus

aureus in all the samples of yoghurt is a source of concern. Its presence in the diary

products is undesirable and should be prevented because it can easily multiply in diary

products if held between 10oC and 45

oC (Atanda and Ikenebomeh, 1991). The

presence of E. coli which is an indicator of faecal contamination and the presence of

other pathogens such as Bacillus, Staphylococcus, and Pseudomonas species indicate

that the yoghurt samples are highly contaminated.

Strains of some fungal genera such as Aspergillus, Fusarium, and Penicillium

reported in this study produce toxins and carcinogenic agents (Uraih and Ogbadu,

2008). Aflatoxin contamination of milk and ice-cream was also reported by Atanda

(2007). Mucor species causes necrosis and thrombosis. The presence of these fungi in

the yoghurt samples also has serious health implications and is of public health

concern as they pose serious health hazards to the unsuspecting consumers.

From the results obtained the microbiological quality of the various yoghurt samples

showed contamination of the samples with different kinds of microorganisms

including potential pathogens which are of public health concern. Proper hygiene and

sanitation therefore should be put in place so as to eradicate these pathogens.

To improve the keeping quality of the yoghurts, the yoghurt should be refrigerated at

about 5°C so as to prevent further production of acid by lactic acid bacteria used in

the production of the yoghurt. It is important that these yoghurts are supplied in

cooling vans other than buses and taxes. The relevant agencies should ensure that

manufacturers of yoghurts follow good manufacturing practices (GMP) guidelines

during and after the production of these products.


(5), 9, 2014 186

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